Steric course of the allylic rearrangement catalyzed by .beta.-hydroxydecanoylthioester dehydrase. Mechanistic implications

Schwab, John M.; Klassen, John B.

doi:10.1021/ja00335a060

Cited by 56 publications

(28 citation statements)

References 4 publications

(4 reference statements)

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“…18,38,39 The first step involves deprotonation of the γ–proton, pro -( R ) in the case of FabA, by a general base (likely the catalytic His) to afford a vinylogous enolate. 40 Until this point, the reaction mechanism mirrors that of a classic isomerase. Instead of suprafacial protonation at the α-position, the subsequent elimination occurs with simultaneous protonation of the δ-hydroxyl group by the catalytic aspartic acid residue, forming the γ,δ-olefin.…”

Section: Discussionmentioning

confidence: 99%

Vinylogous Dehydration by a Polyketide Dehydratase Domain in Curacin Biosynthesis

et al. 2016

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Polyketide synthase (PKS) enzymes continue to hold great promise as synthetic biology platforms for the production of novel therapeutic agents, biofuels and commodity chemicals. Dehydratase (DH) catalytic domains play an important role during polyketide biosynthesis through the dehydration of the nascent polyketide intermediate to provide olefins. Our understanding of the detailed mechanistic and structural underpinning of DH domains that control substrate specificity and selectivity remains limited, thus hindering our efforts to rationally re-engineer PKSs. The curacin pathway houses a rare plurality of possible double bond permutations containing conjugated olefins as well as both cis- and trans-olefins, providing an unrivaled model system for polyketide dehydration. All four DH domains implicated in curacin biosynthesis were characterized in vitro using synthetic substrates and activity was measured by LC-MS/MS analysis. These studies resulted in complete kinetic characterization of the all trans trienoate-forming CurK dehydratase, whose kcat of 72 s−1 is more than three-orders of magnitude greater than any previously reported PKS DH domain. A novel stereospecific mechanism for diene formation involving a vinylogous enolate intermediate is proposed for the CurJ and CurH dehydratases based on incubation studies with truncated substrates. A synthetic substrate was co-crystallized with a catalytically inactive Phe substitution in the His-Asp catalytic dyad of CurJ DH to elucidate substrate-enzyme interactions. The resulting complex suggested the structural basis for dienoate formation and provided the first glimpse into the enzyme-substrate interactions essential for the formation of olefins in polyketide natural products. This examination of both canonical and non-canonical dehydration mechanisms reveals hidden catalytic activity inherent in some DH domains that may be leveraged for future applications in synthetic biology.

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Section: Discussionmentioning

confidence: 99%

Vinylogous Dehydration by a Polyketide Dehydratase Domain in Curacin Biosynthesis

et al. 2016

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“…Once again, this is indicative of a single-base mechanism and not an acid–base mechanism. 51 Notably, it can be observed from the crystal structures (Figure 7) that the catalytic histidine should be able to reach the pro- 4 R proton but not the pro- 4 S proton. This mechanism is outlined in Scheme 4.…”

Section: Dehydratase/hydratase Groupmentioning

confidence: 99%

Active Site Comparisons and Catalytic Mechanisms of the Hot Dog Superfamily

Labonte

Townsend

2012

Chem. Rev.

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“…The mixed anhydride method of Kass and Brock (21) using freshly prepared N-acetylcysteamine (22) gave the NAC thioesters of [1][2][3][4][5][6][7][8][9][10][11][12][13]butyric acid and [~-~~~] h e x a n o i c acid in 70-80% ields. This approach is summarized in Scheme 2.…”

Section: Ef 'mentioning

confidence: 99%

The potential role of fatty acid initiation in the biosynthesis of the fungal aromatic polyketide aflatoxin B₁

Brobst¹,

Townsend²

1994

Can. J. Chem.

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Earlier work in this laboratory has shown the intact incorporation of [1-'3~]hexanoate into averufin (I), a key intermediate in aflatoxin B1 biosynthesis. Parallel experiments with equimolar amounts of [l-13~]butyrate, [l-13~]-3-oxo-octanoate, and [l-13~]-5-oxo-hexanoate gave no detectable specific incorporation of heavy isotope but low and equivalent background incorporation comparable to [l-13~]acetate. Three of these potential intermediates in olyketide formation were reexamined as their P corresponding N-acetylcysteamine (NAC) thioesters. The NAC thioester of [I-' Clhexanoic acid gave a remarkably high 22% intact incorporation while the NAC thioester of [l-13~]-3-oxo-octanoic acid afforded nearly 5% when an equimolar amount was administered to the producing organism Aspergillusparasiticus (ATCC 2455 1). In contrast, the NAC thioester of [~-'~C ] b u t~r i c acid showed no selective enrichment of averufin. This negative result was tested further in a more sensitive experiment with the NAC thioester of [2,3-13~2]butyric acid. No l~c c coupling was detectable, indicatin an incorporation efficiency of <0.1%. F .[ 1 -1 3~, 1 s~2 ]~e x a n o a t e was pre ared and gave a 53% retention of "0 relative to the C lnternal standard in keeping with pre-vious experiments with [l-C, 02]acetate. It is concluded from these data that the initial C6 segment of polyketide biosynthesis is unlikely to arise by P-oxidation of a higher fatty acid but more probably is generated by a specialized fatty acid synthase (FAS) that provides this unit either separately to the polyketide synthase (PKS) or as part of a larger FASIPKS fusion. While these two physical arrangements cannot be distinguished by these experiments, both must accommodate comparatively efficient exchange of the NAC thioesters of both hexanoic and 3-0x0-octanoic acid, but not the NAC thioester of butyric acid. Ef 'tat est en accord avec les expkriences antCrieures avec du [I-13C,l 02]acCtate. Sur la base de ces donnCes on conclut que le segment initial en C6 de la synthkse du polycCtide ne provient probablement pas d'une oxydation P d'un acide gras de poids molCculaire plus ClevC, mais qu'il est vraisemblablement gCnCrC par une synthase d'acide gras spCcialiste (FAS) qui fournit cette unite soit d'une f a~o n sCparCe par rapport B la synthase de polycCtide (PKS) ou dans le cadre d'une fusion plus large FASIPKS. Alors que l'on ne peut pas distinguer entre ces deux arrangements physiques sur la base des expkriences rCalisCes, les deux doivent toutefois accommoder un Cchange efficace des thioesters de la NAC des acides tant hexanoi'que que 3-0x0-octanoi'que, mais pas de l'acide butyrique.[Traduit par la rCdaction]In 1982 we made the unexpected and intriguing observation that [1-13c]hexanoate incorporated label intact into averufin (I), a central intermediate in the biosynthesis of the potent mycotoxin aflatoxin B1 (2). The efficiency of linear fatty acid utilization in these whole-cell experiments was 3 4 % at C-1'. The specific incorporation was seen in a background of c...

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Steric course of the allylic rearrangement catalyzed by .beta.-hydroxydecanoylthioester dehydrase. Mechanistic implications

Cited by 56 publications

References 4 publications

Vinylogous Dehydration by a Polyketide Dehydratase Domain in Curacin Biosynthesis

Vinylogous Dehydration by a Polyketide Dehydratase Domain in Curacin Biosynthesis

Active Site Comparisons and Catalytic Mechanisms of the Hot Dog Superfamily

The potential role of fatty acid initiation in the biosynthesis of the fungal aromatic polyketide aflatoxin B₁

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Steric course of the allylic rearrangement catalyzed by .beta.-hydroxydecanoylthioester dehydrase. Mechanistic implications

Cited by 56 publications

References 4 publications

Vinylogous Dehydration by a Polyketide Dehydratase Domain in Curacin Biosynthesis

Vinylogous Dehydration by a Polyketide Dehydratase Domain in Curacin Biosynthesis

Active Site Comparisons and Catalytic Mechanisms of the Hot Dog Superfamily

The potential role of fatty acid initiation in the biosynthesis of the fungal aromatic polyketide aflatoxin B1

Contact Info

Product

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The potential role of fatty acid initiation in the biosynthesis of the fungal aromatic polyketide aflatoxin B₁